The invention relates to membrane anchor/active compound conjugates having at least one active compound covalently bonded to the membrane anchor compound(s), to a process for their preparation and to their use. The invention further relates to particularly effective foot and mouth disease (FMD) vaccines which can be prepared using membrane-anchoring compounds and certain partial sequences of the FMD virus. Moreover, the invention also relates to a synthetic vaccine containing membrane anchor/active compound conjugates for the specific induction of cytotoxic T-lymphocytes.
Membrane anchor compounds are compounds which can penetrate into biological and synthetic membranes.
For example, these membrane anchor compounds can be natural membrane lipoproteins as have already been isolated from the outer membrane of Escherichia coli and have now also been synthesized. The E. coli membrane anchor compound is composed in the N-terminal region of three fatty acids which are bonded to S-glyceryl-l-cysteine (G. Jung et al. in "Peptides, Structure and Function," W. J. Hruby and D. H. Rich, pages 179 to 182, Pierce Chem. Co. Rockford, Ill., 1983).
Moreover, conformation-stabilized alpha-helical polypeptides have already been described for the investigation of biological membranes by means of models, see inter alia alamethicin, an alpha-helical amphiphilic eicosapeptide antibiotic which forms voltage-dependent ionically conducting systems in lipid membranes (Boheim, G., Hanke, W., Jung, G., Biophys. Struct. Mech. 9, pages 181 to 191 (1983); Schmitt, H. and Jung, G., Liebigs Ann. Chem. pages 321 to 344 and 345 to 364 (1985)).
There is a description in European Patent A1-330 of the immunopotentiating action of lipopeptides which are analogs of the lipoprotein from E. coli which has been known since 1973. Another European patent application, A2-114787, deals with the ability of lipopeptides of this type to activate alveolar macrophages of rats and mice in vitro so that, after incubation with the substance for 24 hours, the macrophages are able to eliminate tumor cells and, in particular, they significantly increase the production of antibodies, for example against porcine serum albumin.
It is proposed in European Patent A2-114787 to use these lipoprotein derivatives as adjuvants for immunization, that is to say to employ the lipoprotein derivatives of the E. coli membrane protein mixed with antigens to improve the immune response.
There is a great need for substances which stimulate and potentiate the immune response, in particular because purified antigens can often be obtained in only minuscule amounts; furthermore, when new batches of antigens are employed there is always the possibility of new contaminants or decomposition products.
It is furthermore desirable not to have to inoculate an experimental animal frequently but, where possible, to obtain the desired immune response by a single dose of the imunogenic material.
Hence it is an object of the present invention to increase the formation of antibodies against antigens or haptens and thus to obtain a specific immunopotentiating action.
According to certain preferred embodiments, the present invention also relates to a vaccine against foot and mouth disease and a process for the preparation thereof.
Foot and mouth disease (FMD) causes great losses in cattle breeding, despite vaccines which have now been available for a long time. One reason for the occurrence of foot and mouth disease at present is the unreliability of classical vaccines which contain killed or inactivated FMD viruses: the inactivation of the virus is occasionally incomplete so that "post-vaccination" outbreaks of FMD may occur (cf. Bohm, Strohmaier, Tierarztl. Umschau 39, 3-8 (1984)). This danger does not exist with synthetic FMD vaccines because in the latter only partial sequences of certain viral proteins, which do not have the function of an intact virus, are used.
Although synthetic FMD vaccines already exist (cf. European Patent Application 0,204,480), they are still in need of improvement.
The present inventors have found that particularly effective FMD vaccines can be prepared using membrane-anchoring compounds and certain partial sequences of the FMD virus. Although the preparation of synthetic vaccines is mentioned in German Offenlegungsschrift DE 3,546,150 A1 as one of many possible uses of membrane anchor/active substance conjugates, it was not to be expected that the conjugates of membrane-anchoring compounds and partial sequences of the FMD virus (membrane anchor/active substance conjugates) would exhibit the exceptional activity which has been found on administration of relatively small amounts of vaccine.
Furthermore, the said vaccines are distinguished, surprisingly, by providing an adequate protection even after a single administration of the vaccine. Moreover, they have the advantage by comparison with conventional vaccines that virtually unlimited storage without cooling is possible.
According to certain preferred embodiments, the invention also relates to a synthetic vaccine for the specific induction of cytotoxic T-lymphocytes.
Cytotoxic T-lymphocytes (killer T-cells) are an essential part of the immune response of warm-blooded animals against intracellular infections. Cytotoxic T-lymphocytes are normally induced only by means of an in vivo vaccination with infectious pathogens (J. Bastin et al., J. Exp. Med., Vol. 165, June 1987). Because of the risks associated with this, a synthetic vaccine for the specific induction of cytotoxic T-lymphocytes would be a substantial improvement. Surprisingly it has now been found that the specific in vivo induction of cytotoxic T-lymphocytes is possible by the use of certain membrane anchor/active compound conjugates containing killer T-cell epitopes.
Although it has been known that membrane anchor/active compound conjugates are suitable for generating neutralizing antibodies (cf. Angew. Chem. 97 (1985), No. 10, p. 883 ff.), a synthetic vaccine containing membrane anchor/active compound conjugates for the specific induction of cytotoxic T-lymphocytes has not yet been reported.
As stated above, it is an object of the present invention to increase the formation of antibodies against antigens or haptens and thus to obtain a specific immunopotentiating action.
The object is achieved according to the invention by the new membrane anchor/active compound conjugate having at least one membrane anchor compound and at least one active compound covalently bonded to the membrane anchor compound(s).
According to the invention, a process for the preparation of membrane anchor compounds is also proposed, which process comprises synthesis of the peptide, which is protected with protective groups in a manner known per se on the functional groups at which no reaction is to take place, by means of known coupling processes on a solid or soluble carrier, such as a polymer (for example Merrifield resin); covalent bonding of the carrier-bound peptides, which have been synthesized in this way, via N-termini or side-groups of the peptide to the membrane anchor compound; isolation of the polymer/peptide conjugate, which has been prepared in this way, by cleavage of the protective groups and the peptide/carrier bond in a manner known per se, and thus the membrane anchor/peptide or the membrane anchor/active compound conjugate being obtained.
The invention also relates to the use of the compounds for the preparation of conventional and monoclonal antibodies in vivo and in vitro; however, it is also possible, in an advantageous manner, to use the compounds according to the invention in genetic engineering to facilitate cell fusion, for the preparation of synthetic vaccines, for the preparation of cell markers with fluorescence labels, spin labels, radioactive labels or the like; for affinity chromatography, in particular for affinity columns; for liposome preparations; as additive to human foodstuffs or animal feeds; and as additive to culture media for microorganisms and, generally, for cell cultures. This may entail, where appropriate, the compounds according to the invention being used, together with vehicles known per se, in solution, ointments, adsorbed onto solid carries, in emulsions or sprays, for purposes in human or veterinary medicine.
According to certain preferred embodiments, the invention also relates to a synthetic vaccine against foot and mouth disease, which vaccine comprises a conjugate of at least one membrane-anchoring compound and at least one partial sequence of a protein of the foot and mouth disease virus.
According to certain preferred embodiments, the invention also relates to a synthetic vaccine for the induction of cytotoxic T-lymphocytes which comprises a conjugate of at least one membrane anchor compound and a protein, containing at least one killer T-cell epitope, of a virus, a bacterium, a parasite or a tumor antigen, or at least one partial sequence containing at least one killer T-cell epitope of a viral, bacterial or parasite protein or of a tumor antigen.